The DEAD-box protein Dbp6 is an ATPase and RNA annealase interacting with the peptidyl transferase center (PTC) of the ribosome

Abstract Ribosomes are ribozymes, hence correct folding of the rRNAs during ribosome biogenesis is crucial to ensure catalytic activity. RNA helicases, which can modulate RNA–RNA and RNA/protein interactions, are proposed to participate in rRNA tridimensional folding. Here, we analyze the biochemical properties of Dbp6, a DEAD-box RNA helicase required for the conversion of the initial 90S pre-ribosomal particle into the first pre-60S particle. We demonstrate that in vitro, Dbp6 shows ATPase as well as annealing and clamping activities negatively regulated by ATP. Mutations in Dbp6 core motifs involved in ATP binding and ATP hydrolysis are lethal and impair Dbp6 ATPase activity but increase its RNA binding and RNA annealing activities. These data suggest that correct regulation of these activities is important for Dbp6 function in vivo. Using in vivo cross-linking (CRAC) experiments, we show that Dbp6 interacts with 25S rRNA sequences located in the 5′ domain I and in the peptidyl transferase center (PTC), and also crosslinks to snoRNAs hybridizing to the immature PTC. We propose that the ATPase and RNA clamping/annealing activities of Dbp6 modulate interactions of snoRNAs with the immature PTC and/or contribute directly to the folding of this region.


Unwinding assays
For the unwinding assay of Fig. S2B, Dbp6 was pre-incubated for 30 min at 30°C with 1 nM of labeled substrate (hemi-duplex ds12/30, i.e. ss30-mer hybridized to radiolabelled ss12-mer) and 50 µM of ATP or ADP, when indicated, in RB 1X (2.5 mM Tris-HCl pH 8, 2.5 mM MgCl2, 10 mM KCl, 0.02 mM DTT, 10 mg/mL BSA) supplemented with 0.03 units of RNasin (Promega). The reaction was pursued for a further 8 min after addition or not of 1 mM ATP. The reaction was then stopped with Stop buffer containing 2 µM of trap oligonucleotide (Comp-58) that is complementary to the ss30-mer and used to prevent reannealing of the radiolabelled ss12-mer oligonucleotide. For the unwinding assay of Fig. S2C, the reaction was carried out as described for Fig. 1D, without pre-incubation. The substrate (1 nM) was a hemi-duplex ds9/30 (ss30-mer hybridized to a radiolabelled ss9-mer).

Ribo Mega-SEC
Ribo Mega-SEC experiments were adapted from (1). Briefly, yeast cells were grown to an OD600nm ~0.6-0.8, incubated with a final concentration of 50 µg/ml of cycloheximide for 10 min, collected by centrifugation followed by two washes with the TMK buffer (20 mM Tris-HCl pH 7.4, 50 mM KCl and 10 mM MgCl2) supplemented with 50 µg/ml of cycloheximide and centrifugation. Yeast pellets were then frozen at -80°C. Cells were thawed and resuspended with extraction buffer (20 mM Hepes-NaOH pH 7.4, 130 mM NaCl, 10 mM MgCl2, 1% CHAPS, 0.2 mg/ml heparin, 2.5 mM DTT, 50 µg/ml cycloheximide and 0.1 units/µl RNasin (Promega)) supplemented with complete EDTA-free protease inhibitor (Roche). Cells were broken by adding two volumes of Zicornia beads and performing 5 runs of 1 min of vigorous vortexing with 1 min of incubation in ice in-between each run. Cell extracts were clarified twice by 10 min centrifugation at 16000xg and quantified by NanoDrop (A260, NP80 Implen).
Aliquots of clarified extracts (50 µl at 2 µg/µl) were injected at a 0.2 ml/min flow rate in a 2000 Å Bio SEC-5 gel filtration column (Agilent) equilibrated with SEC buffer (20 mM Hepes-NaOH pH 7.4, 60 mM NaCl, 10 mM MgCl2, 0.3% CHAPS, 0.2 mg/ml heparin and 2.5 mM DTT). 250 µl fractions were collected that were frozen at -20°C. The samples were then analyzed by western and northern blots to check protein and RNA levels, respectively.  For both gels, lanes 1 and 2 have been loaded with the free 9-mer oligonucleotide and the hemi-duplex, respectively.

Figure S3: Tests associated to the Dbp6 annealing activity
A) Test of ss38-mer self-association. Increasing amounts of biotinylated ss38-mer oligonucleotide (ss38-Biot), as indicated, were mixed for 10 min at 30°C with radiolabelled ss38-mer oligonucleotide (ss38*, 10 nM), either without (lanes 1-4) or with (lanes 5-7) Dbp6 protein (0.8 µM). Lanes 8 and 9 show the migration of the ds38 duplex (ds38*) formed by hybridization of the radioactive ss38-mer oligonucleotide and its complementary oligonucleotide, in the absence or presence of Dbp6. After   Transformed strains were grown in liquid YNB medium supplemented or not with 10 or 100 mM oestradiol. Strains were then serially diluted tenfold, spotted on YNB plates containing or not the indicated concentration of oestradiol and incubated at 30ºC for 2.5 days.   The number of reads (upper graph) and the number of mutations/deletions (lower graph), mapping to each nucleotide of the RDN37 rDNA encoding the 35S pre-rRNA precursor, are plotted for the control (green) and HTP-Dbp6 CRAC (red) experiments. Asterisks indicate common contaminating peaks. A corresponding schematic representation of the pre-rRNA transcript is drawn below, showing the mature 18S (green), 5.8S (light blue) and 25S (dark blue) rRNAs. C) The main Dbp6 crosslinking sites (identified by numbers on Fig. 7) are mapped in blue on the 2D structure of the 25S rRNA. Nucleotides with a high mutation/deletion incidence are highlighted by dark coloured dots within the sequence. The complementary sequences of the major snoRNAs bound by Dbp6 (listed in Fig. 7) are indicated in red.

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